RESUMO
Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.
Assuntos
Doença de Chagas/tratamento farmacológico , Modelos Animais de Doenças , Inflamação/prevenção & controle , Fígado/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Nitroimidazóis/farmacologia , Sepse/tratamento farmacológico , Tripanossomicidas/farmacologia , Animais , Antioxidantes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Nitroimidazóis/uso terapêutico , Estresse Oxidativo , Receptor 4 Toll-Like/metabolismo , Tripanossomicidas/uso terapêuticoRESUMO
Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200µM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3h of exposure, returning to normality at 24h. Additionally, BZL increased glutathione peroxidase activity at 12h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos , Fator 2 Relacionado a NF-E2 , Nitroimidazóis , Estresse Oxidativo , Tripanossomicidas , Animais , Humanos , Masculino , Camundongos , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Células Hep G2 , Camundongos Endogâmicos C57BL , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Nitroimidazóis/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , RNA Interferente Pequeno/efeitos dos fármacos , Tripanossomicidas/farmacologiaRESUMO
CONTEXT: We have previously reported that benznidazole (BZL), known for its trypanocidal action, has anti-proliferative activity against different cell lines like HeLa and Raw 264.7 among others. At the moment, it has not been reported if the anti-proliferative effect of BZL is similar for non-adherent hematopoietic cells like was reported for adherent cancer cell lines. OBJECTIVE: We aimed to investigate the efficacy of BZL on the growth of the leukemic cell lines THP-1 and OCI/AML3. MATERIALS AND METHODS: We evaluated cell proliferation by [³H]-thymidine incorporation and MTT reduction as well as cell death by lactate dehydrogenase (LDH) activity. We assessed apoptosis by flow cytometry for detection of annexin V-positive and propidium iodide-negative cells, along with nuclear morphology by diamidino-2-phenolindole (DAPI) staining. Western blot studies were performed to evaluate changes in cell cycle proteins in BZL-treated cells. RESULTS: BZL significantly reduced proliferation of both cell lines without inducing cell death. Likewise it produced no significant differences in apoptosis between treated cells and controls. In addition, flow cytometry analysis indicated that BZL caused a larger number of THP-1 cells in G0/G1 phase and a smaller number of cells in S phase than controls. This was accompanied with an increase in the expression of the CDK inhibitor p27 and of cyclin D1, with no significant differences in the protein levels of CDK1, CDK2, CDK4, cyclins E, A and B as compared to controls. CONCLUSION: BZL inhibits the proliferation of leukemic non-adherent cells by controlling cell cycle at G0/G1 cell phase through up-regulation of p27.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Leucemia Monocítica Aguda/metabolismo , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Morte Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/biossíntese , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Leucemia Monocítica Aguda/patologia , Proteínas de Neoplasias/biossínteseRESUMO
Interferon-gamma/transforming growth factor-beta (IFN-gamma/TGF-beta) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-alpha/TGF-beta. In previous studies, we showed that IFN-alpha2b increases TGF-beta(1) production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-alpha/TGF-beta(1) pathways was explored. We observed a positive cross-talk between IFN-alpha and TGF-beta(1) signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-beta(1) and IFN-alpha signals. Interestingly, we reported the novel finding that the autocrine TGF-beta(1) up-regulates TGF-betaRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-alpha2b and by autocrine TGF-beta(1) are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Interferon-alfa/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Interferon alfa-2 , Fígado/citologia , Fígado/metabolismo , Fígado/fisiopatologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Masculino , Lesões Pré-Cancerosas/fisiopatologia , Ratos , Ratos Wistar , Proteínas Recombinantes , Fator de Transcrição STAT1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Leucotrieno B4/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Ciclina D1/metabolismo , Eicosanoides/metabolismo , Hepatectomia/métodos , Concentração de Íons de Hidrogênio , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Leucotrienos/metabolismo , Fígado/efeitos dos fármacos , Regeneração Hepática/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Islet-Neogenesis Associated Protein-Pentadecapeptide (INGAP-PP) increases ß-cell mass and enhances glucose and amino acids-induced insulin secretion. Our aim was to demonstrate its effect on liver metabolism. For that purpose, adult male Wistar rats were injected twice-daily (10â¯days) with saline solution or INGAP-PP (250⯵g). Thereafter, serum glucose, triglyceride and insulin levels were measured and homeostasis model assessment (HOMA-IR) and hepatic insulin sensitivity (HIS) were determined. Liver glucokinase and glucose-6-phosphatase (G-6-Pase) expression and activity, phosphoenolpyruvate carboxykinase (PEPCK) expression, phosphofructokinase-2 (PFK-2) protein content, P-Akt/Akt and glycogen synthase kinase-3ß (P-GSK3/GSK3) protein ratios and glycogen deposit were also determined. Additionally, glucokinase activity and G-6-Pase and PEPCK gene expression were also determined in isolated hepatocytes from normal rats incubated with INGAP-PP (5⯵g/ml). INGAP-PP administration did not modify any of the serum parameters tested but significantly increased activity of liver glucokinase and the protein level of its cytosolic activator, PFK-2. Conversely, INGAP-PP treated rats decreased gene expression and enzyme activity of gluconeogenic enzymes, G-6-Pase and PEPCK. They also showed a higher glycogen deposit and P-GSK3/GSK3 and P-Akt/Akt ratio. In isolated hepatocytes, INGAP-PP increased GK activity and decreased G-6-Pase and PEPCK expression. These results demonstrate a direct effect of INGAP-PP on the liver acting through P-Akt signaling pathway. INGAP-PP enhances liver glucose metabolism and deposit and reduces its production/output, thereby contributing to maintain normal glucose homeostasis. These results reinforce the concept that INGAP-PP might become a useful tool to treat people with impaired islet/liver glucose metabolism as it occurs in T2D.
Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Fígado/metabolismo , Oligopeptídeos/farmacologia , Proteínas Associadas a Pancreatite/química , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Oligopeptídeos/química , Ratos , Ratos WistarRESUMO
Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose/genética , Insulina/metabolismo , Resistência à Insulina , Interleucina-1beta/metabolismo , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de SinaisRESUMO
Interferon-alpha2b (IFN-alpha2b) is an important component in the preventive treatment of patients who have severe hepatic illness such as hepatitis B or C and hepatocarcinomas. In a previous work, using a rat liver preneoplastic model, we have demonstrated that IFN-alpha2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-beta(1). In this study, the implication of hepatocytes redox status of IFN-alpha2b-treated preneoplastic liver in the TGF-beta(1)-induced apoptotic death was analyzed. Results indicate that IFN-alpha2b induces hepatocytic TGF-beta(1) production and secretion by induction of reactive oxygen species (ROS) formation through the activation of a membrane bound NADPH oxidase complex. TGF-beta(1), in turn, reduces hepatocytes antioxidant defenses and induces programmed cell death. On the other hand, it was also demonstrated that treatment of rats with IFN-alpha2b plus a ROS scavenger such as ascorbic acid, abolishes the apoptotic effect of IFN-alpha2b in rat preneoplastic livers, leading to an increase of the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling and/or regulator molecules in the IFN-alpha2b-induced apoptosis in hepatic preneoplastic cells.
Assuntos
Apoptose/efeitos dos fármacos , Interferon-alfa/farmacologia , Lesões Pré-Cancerosas/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/fisiologia , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Interferon alfa-2 , Fígado/citologia , Masculino , NADPH Oxidases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.
Assuntos
Regeneração Hepática/fisiologia , Fígado , Neovascularização Fisiológica/fisiologia , Óxido Nítrico/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Western Blotting , Espectroscopia de Ressonância de Spin Eletrônica , Fígado/irrigação sanguínea , Fígado/metabolismo , Fígado/fisiologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Nitratos/metabolismo , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Nitroprussiato/farmacologia , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Ligaria cuneifolia (R et P) Tiegh. (Loranthaceae) (Lc) aqueous extract-treated rats by via intraperitoneal (i.p.) show increased blood viscosity and decreased plasma cholesterol (Chol) levels. In this work, we analize the effect of the vehicle polyvinylpyrrolidone (PVP) and that of the Methanolic Fraction of the extract of Lc (MFLc) on hemorrheological properties in vivo and in vitro and on biliary excretion. For in vivo conditions, adult male Wistar rats were divided in five experimental groups (n=5 each one) which were injected, every 24 hr during 3 days by via i.p., with: (1) saline solution (Control); (2) PVP 0.47 mg/100 g bw; (3) MFLc 0.95 mg/100 g bw plus PVP 0.47 mg/100 g bw; (4) PVP 12.5 mg/100 g bw; and (5) MFLc 23.0 mg/100 g bw plus PVP 12.5 mg/100 g bw. Intended for in vitro conditions, blood samples obtained by heart puncture were divided into three fractions, which were incubated with: saline solution (Control), PVP 12.5 mg%, and MFLc 25 mg% plus PVP 12.5 mg%. We demonstrated a direct effect of PVP alone and of MFLc "per se" on the erythrocyte membrane resulting in a cell shape change from dyscocyte to spherostomatocyte (MI more negative) as well as a decrease in erythrocyte deformability (increased RI). These changes induce an increase in blood viscosity. Decreased plasma Chol is a consequence of an increased bile salts biliary excretion.
Assuntos
Viscosidade Sanguínea/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Loranthaceae , Extratos Vegetais/farmacologia , Povidona/farmacologia , Animais , Bile/efeitos dos fármacos , Colesterol/sangue , Sistemas de Liberação de Medicamentos , Masculino , Folhas de Planta , Ratos , Ratos WistarRESUMO
We analysed the possible cellular mechanism involved in the NO action in the balance between apoptosis and cell proliferation in liver regeneration process. We determined p53, proapoptotic protein Bax, antiapoptotic Bcl-xL, proliferating cell nuclear antigen (PCNA) and apoptotic index at the early stages of regenerative process after NO increase by lipopolysaccharide-induction (LPS) of inducible-type nitric oxide synthase (iNOS) and by direct NO donor (sodium nitroprusside, SNP). Male Wistar rats were randomised in four experimental groups: sham operated control (Sh), partial hepatectomised control (PH-C), partial hepatectomised pretreated with LPS (2 mg/kg body weight, i.p.) (PH-LPS), and partial hepatectomised pretreated with SNP (2.5 mg/kg body weight, i.v. at a rate of 1 ml/h) (PH-SNP). Animals were killed 5 h post-surgery. Hepatic cytosolic iNOS showed an increase of 34% in PH-C animals with respect to Sh, and LPS-treatment increased iNOS protein levels 30% compared with PH-C. Bax and p53 protein levels showed significant increases in LPS- and SNP-treated hepatectomised rats with respect to PH-C. The apoptotic indexes were increased 75% in both, PH-LPS and PH-SNP rats versus PH-C. The increase of NO did not show any change in the proliferation process. These results suggest that NO is involved in apoptosis via p53 and Bax proteins after PH, showing a tightly regulated growth process in liver regeneration.
Assuntos
Apoptose , Regeneração Hepática , Óxido Nítrico/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Hepatectomia , Masculino , Óxido Nítrico Sintase/farmacologia , Óxido Nítrico Sintase Tipo II , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Wistar , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2RESUMO
UNLABELLED: It was demonstrated that Ligaria cuneifolia (Lc) crude extract increased blood viscosity and decreased plasma cholesterol in rats. In the present study, we analyzed the Lc proanthocyanidin enriched fraction (PLc) to determine if it is capable of altering the hemorheological parameters while diminishing the plasma cholesterol. In vivo studies in adult male Wistar rats, randomized in three groups (nâ=â6 each one) were performed: 1. CONTROL: saline intraperitoneal (i.p.); 2. PLc 0.6âmg/100âg body weight (b.w.) i.p. and 3. PLc 3âmg/100âg b.w. i.p., every 24 hours during 3 days. IN VITRO STUDIES: with blood obtained by cardiac puncture, separated in aliquots and incubated with: 1. Saline solution (Control); 2. PLc 0.1âmg/mL, and 3. PLc 1.0âmg/mL, equivalent to doses in vivo experiments. The results demonstrated that in vivo PLc 0.6 and PLc 3 reduced plasma cholesterol (Cho) and LDL-Cho. Neither blood nor plasma viscosity was altered. Decrease of plasma cholesterol could be due to an increase of cholesterol and bile salts excretion leading to an increase of bile flow. In vitro experiments showed a direct interaction of PLc, at high concentration, with the erythrocyte membrane, inducing a switch from discocyte to stomatocyte. Only, PLc without hepatic metabolism produces hemorheological changes. Thus, PLc in vivo might be a pharmacological agent capable of decreasing plasma cholesterol.
Assuntos
Colesterol/sangue , Hemorreologia/efeitos dos fármacos , Loranthaceae/química , Extratos Vegetais/química , Folhas de Planta/química , Animais , Viscosidade Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Masculino , Extratos Vegetais/farmacologia , Proantocianidinas , Ratos , Ratos WistarRESUMO
Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) (Argentine mistletoe) is usually used in local folk medicine. This work focuses on the hemorrheologic parameters in the treatment with an aqueous extract of Ligaria cuneifolia (Lc) by two different administration routes: intraperitoneal (i.p.) and intravenous (i.v.). Adult male Wistar rats were injected by via i.p. or by via i.v. with: saline solution; 2.5 mg/100 g body weight of Lc and 5.5 mg/100 g body weight of Lc. The relative viscosity of blood (eta r)(45/Hct) was measured showing that Lc-treatment by via i.p. produced an increase of about 69% while Lc by via i.v. enhanced the parameter about 47%. All of Lc-treated animals showed a significant increase in the rigidity index (RI). The mean corpuscular hemoglobin concentration (MCHC) exhibited an increase of about 15% in all the treated groups. Lc-treatment by via i.p. produced a diminution of plasma cholesterol level associated with RI augmentation which induced an increase of (eta r)(45/Hct). By via i.v. Lc produces both RI and (eta r)(45/Hct) augmentation by increasing MCHC but without modifying plasma cholesterol level, indicating a direct Lc-action on the internal viscosity of the erythrocyte.
Assuntos
Hemorreologia/efeitos dos fármacos , Loranthaceae/química , Extratos Vegetais/farmacologia , Animais , Argentina , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Viscosidade Sanguínea/efeitos dos fármacos , Colagogos e Coleréticos/farmacologia , Colesterol/sangue , Avaliação Pré-Clínica de Medicamentos , Índices de Eritrócitos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Injeções Intraperitoneais , Injeções Intravenosas , Fígado/efeitos dos fármacos , Masculino , Medicina Tradicional , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , ÁguaRESUMO
SCOPE: Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G1 and S phases, accumulation in G2, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION: The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.
Assuntos
Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Quercetina/farmacologia , Animais , Proteína Quinase CDC2/metabolismo , Divisão Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , PPAR alfa/metabolismo , Ratos , Ratos WistarRESUMO
Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP. Our results show that BZL reduces leukocytes in peripheral blood accompanied by an increase in peritoneal macrophages 24h after CLP. In the liver of these septic mice, BZL decreased expression of mRNA and protein for TNF-α and NOS-2 by inhibition of NF-κB and MAPK (p-38 and ERK). The body of evidence suggests that the immunomodulatory effects of BZL could act selectively, as it is able to decrease the systemic inflammatory reaction and the hepatic response but it can increase the number of cells in the site of infection.
Assuntos
Ceco/patologia , Fígado/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Nitroimidazóis/uso terapêutico , Sepse/tratamento farmacológico , Sepse/enzimologia , Animais , Ceco/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Contagem de Leucócitos , Ligadura , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitroimidazóis/farmacologia , Punções , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sepse/fisiopatologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
UNLABELLED: We tested the in vivo and the in vitro effects of both Ligaria cuneifolia catechin- and quercetin-enriched fractions on erythrocyte shape and deformability, and on plasma cholesterol level. For in vivo studies, adult male Wistar rats were randomized in three experimental groups which received intraperitoneally, once a day, 3 days: CONTROL: saline solution (C; n = 6); catechin from L. cuneifolia, 0.60 mg/100 g body weight (CLc; n = 6), or quercetin from L. cuneifolia, 2.3 mg/100 g body weight (QLc; n = 6). For in vitro studies, blood samples obtained from male Wistar rats were divided into three fractions, which were incubated with saline solution (C), catechin (CLc; n = 5) and quercetin (QLc; n = 5), in a concentration equivalent to 0.60 mg/100 g body weight, and 2.3 mg/100 g body weight, respectively. CLc significantly reduced the rigidity index due to a diminished mean concentration volume. QLc induced erythrocyte rigidization (less deformability), thus increasing blood viscosity. Neither of the two treatments produced any changes in plasmatic or biliary excretion of cholesterol. Opposite results were observed in rigidity index with CLc and QLc. In vitro studies showed an interaction of both CLc and QLc with the erythrocyte membrane, which induced changes in the erythrocyte shape from discocyte to stomatocyte.
Assuntos
Viscosidade Sanguínea/efeitos dos fármacos , Catequina/farmacologia , Colesterol/sangue , Loranthaceae/química , Extratos Vegetais/farmacologia , Quercetina/farmacologia , Animais , Catequina/química , Forma Celular/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemorreologia/efeitos dos fármacos , Masculino , Quercetina/química , Ratos , Ratos WistarRESUMO
Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.
Assuntos
Diuréticos/toxicidade , Ácido Etacrínico/toxicidade , Glutationa Transferase/metabolismo , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , 2-Acetilaminofluoreno/toxicidade , Transportadores de Cassetes de Ligação de ATP/metabolismo , Alquilantes/toxicidade , Animais , Carcinógenos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dietilnitrosamina/toxicidade , Resistência a Medicamentos , Glutationa/metabolismo , Hepatócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.
Assuntos
Apoptose , Hepatócitos/citologia , Interferon-alfa/farmacologia , Neoplasias Hepáticas/metabolismo , Fígado/citologia , Lesões Pré-Cancerosas/metabolismo , Animais , Anexina A5/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA , Hepatócitos/metabolismo , Interferon alfa-2 , Masculino , Potencial da Membrana Mitocondrial , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes , Fator de Crescimento Transformador beta1/metabolismoRESUMO
In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.
Assuntos
Apoptose , Interferon-alfa/farmacologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Lesões Pré-Cancerosas/fisiopatologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultura/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Interferon alfa-2 , Fígado/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes , Proteína Smad2 , Proteína Smad3 , Transativadores/metabolismo , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta1RESUMO
BACKGROUND: This work aims to investigate the role of lipid peroxidation (LPO) at early stages of liver regeneration and to evaluate the balance between apoptosis and cell proliferation during this process. METHODS: Sham and partial hepatectomized (PH) male Wistar rats were randomized in seven groups: Control (untreated), E-Control (injected with vitamin E-vehicle), C-Control (injected with vitamin C-vehicle), E1 (vitamin E 100 mg/kg body weight), E2 (vitamin E 600 mg/kg body weight), C1 (vitamin C 30 mg/kg body weight), C2 (vitamin C 100 mg/kg body weight). RESULTS: Vitamin treatments attenuated the increase of LPO level observed in total homogenate and microsomes at 3 and 5 hr after PH. Both antioxidant vitamins attenuated the increase in Bax pro-apoptotic protein and augmented Bcl-xL antiapoptotic protein levels (35%) at 3 and 5 hr post-PH; Bcl-xL/Bax ratio was, therefore, increased. A direct linear relationship between LPO levels and Bax mitochondrial protein levels was seen. Vitamin-treatments diminished the apoptosis index with respect to PH-Control values, so that this parameter showed a linear relationship with LPO levels. At 24 hr after PH, the vitamin treatments increased the peak of [(3) H]-thymidine incorporation into DNA and the proliferative index (PI), measured as PCNA expression; an inverse relationship between PI and LPO levels could be demonstrated. CONCLUSION: Our data show that the diminution of LPO levels by vitamin-treatment post-PH produces both an attenuation of cellular apoptosis and a marked increase in the proliferation process, suggesting that the modulation of LPO has a role in liver regeneration process.